DESCRIPTION: This application proposes to identify and phenotypically characterize mutants affecting segmentation in the parasitic wasp, Nasonia vitripennis. The long-term goal of this project is to compare the segmentation process in Drosophila and Nasonia in order to determine whether the two regulatory hierarchies are conserved with respect to overall structure, functional classes, and individual genes. Many of the genes identified in the Drosophila segmentation hierarchy are conserved throughout metazoans, and in some cases, significant features of the expression patterns are conserved as well. However, the functional similarities that might be inferred have rarely been tested, due to the lack of available mutations in most species. The importance of combining both genetic and molecular analysis is suggested by cases where there is a lack of congruence between expression patterns and genetically defined functions. In this proposal, the two approaches will be combined by using mutations generated in the wasp together with molecular probes for Drosophila genes which readily cross-react with apparent homologues in the wasp. Three specific aims are proposed. First, the wasp genome will be screened for recessive, EMS-induced zygotic lethal mutations which disrupt the embryonic segmentation pattern. The screen exploits the ability of unfertilized female wasps to generate large numbers of haploid males, such that the entire genome (which consists of five chromosomes) can be screened in a single experiment. For the screen, F1 females will be generated by mating EMS-treated males with appropriately marked females. The hatching frequency of male progeny from individual unmated F1 females will be determined, and recessive embryonic lethals which are fully penetrant will be inferred in cases where approximately 50% of the embryos fail to hatch. To allow recovery of temperature- sensitive mutations, the F1 females will be reared at 18 degrees and the F2 progeny assayed at 28 degrees. Unhatched embryos from lethal lines will be cleared and scored for visible phenotypes. Mothers whose broods show interesting pattern defects will be mated to allow recovery of the mutation in F2 females. In subsequent generations, conventional linkage analysis will be used to map mutations with respect to a small number of recessive visible markers. Long-term stocks will be preserved in the form of diapause larvae, which are induced by maintaining mothers in a cool, dark environment; these remain quiescent for periods from 4-18 months. Allelism between independently-isolated mutations will be inferred by two criteria: similarity of phenotype and similarity of genetic map position. In this system, the inability to carry recessive lethals in haploid males precludes simple complementation tests. The screen will be continued until the genome appears to be half saturated, based upon the partitioning of newly induced mutations between previously identified and novel loci. Depending on the outcome of further phenotypic analyses, screening might be resumed at a later time in an attempt to approach saturation. Mutants identified in the screen will be examined morphologically. Cuticular patterns will be used to determine which regions of the embryo require gene function, and loci will be categorized to determine whether the familiar Drosophila classes of gap, pair-rule, and segment polarity genes are represented in Nasonia. The embryonic development of selected mutants will be followed with time-lapse video microscopy. Finally, selected mutants will be analyzed by antibody staining with selected probes representing members of the Drosophila segmentation hierarchy, with an initial focus on the pattern of engrailed stripes. In cases where the engrailed pattern is altered in a manner reminiscent of particular segmentation mutants in Drosophila, additional probes whose regulation in Drosophila is well-characterized will be tested.